Viral infectious diseases are major public healthcare issues. Human Hepatitis B virus (HBV) is a member of a family of DNA viruses that primarily infect the liver (Gust, 1986). Other members of this family are woodchuck hepatitis B virus (WHV) ((Summers, Smolec et al. 1978), duck hepatitis B virus (DHBV) ((Mason, Seal et al. 1980) and heron hepatitis B virus (HHBV) (Sprengel, Kaleta et al. 1988). These viruses share a common morphology and replication mechanisms, but are species specific for infectivity (Marion, 1988).
HBV primarily infects liver cells and can cause acute and chronic liver disease resulting in cirrhosis and hepatocellular carcinoma. Infection occurs through blood and other body fluids. Approximately 90% of the individuals infected by HBV are able to clear the infection, while the remaining 10% become chronic carriers of the virus with a high probability of developing cirrhosis of the liver and hepatocellular carcinoma. The world Health Organization statistics show that more than 2 billion people have been infected by HBV and among these, 350 million are chronically infected by the virus (Beasley 1988) (Law J Y 1993). Prophylactic vaccines based on HBV surface antigen (HbSAg) have been very effective in providing protective immunity against HBV infections. These vaccines have been developed from HbSAg purified from plasma of chronic HBV carriers, produced by recombinant DNA techniques as well as through the use of synthetic peptides (Please see U.S. Pat. Nos. 4,599,230 and 4,599,231). These vaccines are highly effective in the prevention of infection, but are ineffective in eradicating established chronic infections.
Human Hepatitis B Virus (HBV) belongs to the family of Hepadnaviruses. Other members of this family are Duck Hepatitis B Virus (DHBV), Woodchuk Hepatitis Virus (WHV) Ground squirrel Hepatitis B Virus (GSHV) and Heron Hepatitis B Virus (HHBV). Although these viruses have similar morphology and replication mechanism, they are fairly species specific consequently, infect only very closely related species. These viruses have a DNA genome ranging in size of 3.0-3.2 Kb, with overlapping reading frames to encode several proteins. HBV genome encodes several proteins. Among these, the surface antigens: Large (S1/S2/S), Medium (S2/S) and Small (S) are proposed to be involved in the binding of the virus to the cellular receptors for uptake. The core protein (Core) forms capsids which encapsulate the partially double stranded DNA genome. Polymerase/Reverse Transcriptase (Pol) protein is a multifunctional enzyme necessary for the replication of the virus. The X protein has been proposed to have many properties, including the activation of Src kinases (Ganem and Schneider, 2001). The present invention describes DNA sequences and amino acid compositions of the surface antigen proteins S1/S2, S1/S2/S as well as Core protein fusion proteins with a xenotypic Mab fragment.
DHBV, another member of the hepdnaviral family, infects pekin ducks, are species specific, and have served as an animal model for studying the hepatitis B viruses. DHBV has a DNA genome and it codes for surface antigens PreS and PreS/S, Core protein (Core) and Polymerase/Reverse Transcriptase. The present invention also describes DNA sequences and deduced amino acid sequences of fusion proteins of the PreS, PreS/S and Core proteins with a fragment of a xenotypic Mab. These fusion proteins can be used to elicit a broad immune response in chronic viral infections, thus as therapeutic vaccine.
Hepatitis C virus (HCV) is a member of the flaviviridae family of RNA viruses. Route of infection is via blood and body fluids and over 50% of the patients become chronic carriers of the virus. Persistent infection result in chronic active hepatitis which may lead to liver cirrhosis and hepatocellular carcinoma (Saito et. al. (1990) PNAS USA 87: 6547-6549).
Approximately 170 million people worldwide are chronic carriers of HCV (Wild & Hall (2000) Mutation Res. 462: 381-393). There is no prophylactic vaccine available at present. Current therapy is Interferon α2b and Ribavirin, either alone or as combination therapy. The significant side effects for interferon treatment and the development of mutant strains are major drawbacks to the current therapy. Moreover, interferon therapy is effective only in 20% of the patients. Therapeutic vaccines to enhance host immune system to eliminate chronic HCV infection will be a major advancement in the treatment of this disease.
HCV genome is a positive sense single stranded RNA molecule of approximately 9.5 Kb in length. This RNA which contains both 5′ and 3′ untranslated regions that code for a single polyprotein which is cleaved into individual proteins and catalyzed by both viral and host proteases (Clarke, B. (1997) J. Gen. Virol. 78: 2397-2410). The structural proteins are Core, Envelope E1 & E2 and P7. The non-structural proteins are NS2, NS3, NS4A, NS4B, NS5A and NS5B. Core forms capsids. E1, E2 are envelope proteins, also called “Hypervariable region” due to the high rate of mutations. NS3 is a Serine Protease, the target of several protease inhibitors as antivirals for HCV. NS5B is the RNA Polymerase enzyme. NS5A has recently been suggested to have a direct role in the replication of the virus in the host by counteracting the interferon response Tan, S-L & Katze, M. G. (2001) Virology 284: 1-12) which augments the immune function.
When a healthy host (human or animal) encounters an antigen (such as proteins derived from a bacterium, virus and/or parasite), normally the host initiates an immune response. This immune response can be a humoral response and/or a cellular response. In the humoral response, antibodies are produced by B-cells and are secreted into the blood and/or lymph in response to an antigenic stimulus. The antibody then neutralizes the antigen, e.g. a virus, by binding specifically to antigens on its surface, marking it for destruction by phagocytotic cells and/or complement-mediated mechanisms. The cellular response is characterized by the selection and expansion of specific helper and cytotoxic T-lymphocytes capable of directly eliminating the cells which contain the antigen.
In many individuals, the immune system does not respond to certain antigens. When an antigen does not stimulate the production of a specific antibody and/or killer T-cells, the immune system is unable to prevent the resultant disease. As a result, the infectious agent, e.g. virus, can establish a chronic infection and the host immune system becomes tolerant to the antigens produced by the virus. The mechanism by which the virus evades the host immune machinery is not clearly established. The best-known examples of chronic viral infections include Hepatitis B, Hepatitis C, Human Immunodeficiency Virus and Herpes Simplex Virus.
In chronic states of viral infections, the virus escapes the host immune system. Viral antigens are recognized as “self,” and thus not recognized by the antigen-presenting cells. The lack of proper presentation of the appropriate viral antigen to the host immune system may be a contributing factor. The success in eliminating the virus will result from the manner in which the antigen is processed and presented by the antigen presenting cells (APCs) and the involvement of the regulatory and cytotoxic T cells. The major participant in this process is the Dendritic Cell (DC), which captures and processes antigens, expresses lymphocyte co-stimulatory molecules, migrates to lymphoid organs, and secretes cytokines to initiate immune responses. Dendritic cells also control the proliferation of B and T lymphocytes which are the mediators of immunity (Steinman et al 1999). The generation of a cytotoxic T cell (CTL) response is critical in the elimination of the virus infected cells and thus a cure of the infection.
Antigen Presenting Cells process the encountered antigens differently depending on the localization of the antigen (Steinman et al 1999). Exogenous antigens are processed within the endosomes of the APC and the generated peptide fragments are presented on the surface of the cell complexed with Major Histocompatibility Complex (MHC) Class II. The presentation of this complex to CD4+ T cells stimulate the CD4+ T helper cells. As a result, cytokines secreted by the helper cells stimulate B cells to produce antibodies against the exogenous antigen (humoral response). Immunizations using antigens typically generate antibody response through this endosomal antigen processing pathway.
On the other hand, intracellular antigens are processed in the proteasome and the resulting peptide fragments are presented as complexes with MHC Class I on the surface of APCs. Following binding of this complex to the co-receptor CD8 molecule, antigen presentation to CD8+ T cells occurs which result in cytotoxic T cell (CTL) immune response to remove the host cells that carry the antigen.
In patients with chronic viral infections, since the virus is actively replicating, viral antigens will be produced within the host cell. Secreted antigens will be present in the circulation. As an example, in the case of chronic HBV carriers, virions, the HBV surface antigens and the core antigens can be detected in the blood. An effective therapeutic vaccine should be able to induce strong CTL responses against an intracellular antigen or an antigen delivered into the appropriate cellular compartment so as to activate the MHC Class I processing pathway.
These findings would suggest that a therapeutic vaccine that can induce a strong CTL response should be processed through the proteasomal pathway and presented via the MHC Class I (Larsson, Fonteneau et al. 2001). This can be achieved either by producing the antigen within the host cell, or it can be delivered to the appropriate cellular compartment so that it gets processed and presented so as to elicit a cellular response. Several approaches have been documented in the literature for the intracellular delivery of the antigen. Among these, viral vectors ((Lorenz, Kantor et al. 1999), the use of cDNA-transfected cells (Donnelly, Ulmer et al. 1997) (Donnelly et al 1997) as well as the expression of the antigen through injected cDNA vectors (Lai and Bennett 1998) (U.S. Pat. No. 5,589,466), have been documented.
Delivery vehicles capable of carrying the antigens to the cytosolic compartment of the cell for MHC Class I pathway processing have also been used. The use of adjuvants to achieve the same goal has been described in detail by (Hilgers et al. 1999) Another approach is the use of biodegradable microspheres in the cytoplasmic delivery of antigens (Newman, Kwon et al. 2000), exemplified by the generation of a Th1 immune response against ovalbumin peptide (Newman, Samuel et al. 1998; Newman, Kwon et al. 2000). It has also been shown that PLGA nanospheres are taken up by the most potent antigen presenting cells, dendritic cells (Newman, Elamanchili et al. 2002).
Dendritic cells derived from blood monocytes, by virtue of their capability as professional antigen presenting cells have been shown to have great potential as immune modulators which stimulate primary T cell response (Steinman, Inaba et al. 1999), (Banchereau and Steinman 1998). This unique property of the DCs to capture, process, present the antigen and stimulate naïve T cells has made them very important tools for therapeutic vaccine development (Laupeze, Fardel et al. 1999). Targeting of the antigen to the DCs is the crucial step in the antigen presentation and the presence of several receptors on the DCs for the Fc region of monoclonal antibodies have been exploited for this purpose (Regnault, Lankar et al. 1999). Examples of this approach include ovarian cancer Mab-B43.13, Anti-PSA antibody as well as Anti-HBV antibody antigen complexes (Wen, Qu et al. 1999). Cancer immunotherapy using DCs loaded with tumor associated antigens have been shown to produce tumor-specific immune responses and anti-tumor activity (Campton, Ding et al. 2000; Fong and Engleman 2000). Promising results were obtained in clinical trials in vivo using tumor-antigen-pulsed DCs (Tarte and Klein 1999). These studies clearly demonstrate the efficacy of using DCs to generate immune responses against cancer antigens.